DNA Of Higher Eukaryotes Research Articles

Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation.

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.

Synthesis of RNA Containing 5-Hydroxymethyl-, 5-Formyl-, and 5-Carboxycytidine.

5-Hydroxymethyl-, 5-formyl-, and 5-carboxy-2'-deoxycytidine are new epigenetic bases (hmdC, fdC, cadC) that were recently discovered in the DNA of higher eukaryotes. The same bases (5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine; hmC, fC, and caC) have now also been detected in mammalian RNA with a high abundance in mRNA. While DNA phosphoramidites (PAs) that allow the synthesis of xdC-containing oligonucleotides for deeper biological studies are available, the corresponding silyl-protected RNA PAs for fC and caC have not yet been disclosed. Here, we report novel RNA PAs for hmC, fC, and caC that can be used in routine RNA synthesis. The new building blocks are compatible with the canonical PAs and also with themselves, which enables even the synthesis of RNA strands containing all three of these bases. The study will pave the way for detailed physical, biochemical, and biological studies to unravel the function of these non-canonical modifications in RNA.

Arginine CGA codons as a source of nonsense mutations: a possible role in multivariant gene expression, control of mRNA quality, and aging.

Methylation of cytosine residues in DNA of higher eukaryotes, including humans, creates "hot spots" of C→T transitions in the genome. The predominantly methylated sequence in mammalian DNAs is CG (CpG). Among CG-containing codons, CGA codons for arginine are unique due to their ability to create stop codons TGA (UGA in mRNA) upon epigenetic-mediated mutation. As such nonsense mutations can have a strong adverse effect on the cell and organism, we have performed a study, on the example of human genes, aimed to characterise the anticipated effects of epigenetic-mediated nonsense mutations CGA→TGA in somatic cells. It is commonly accepted that premature termination codons (PTCs) lead to the biosynthesis of truncated and usually inactive proteins. In addition, transcripts with PTC can be destroyed by a nonsense-mediated mRNA decay (NMD) machinery. We have considered the cell potentialities (gene families, diploidy, and alternative splicing) to overcome the worst consequences of nonsense mutation. As a special case, in the biosynthesis of a particular group of proteins called selenoproteins, the mutation CGA→UGA would not lead to the premature translation termination and NMD but rather to the insertion of selenocysteine or cysteine instead of former arginine. The finding of SECIS (Sel insertion sequence)-like structures in a variety of mRNAs allowed us to postulate the existence of facultative selenoproteins, whose biosynthesis might be coupled with the redefinition of premature UGA stop codons arising upon mutations, as in the case of "classic" selenoproteins. Nevertheless, a detailed structural analysis of 165 transcripts has shown that roughly 80-90% of functional human mRNAs are potential substrates for NMD upon the PTC emergence. A hypothesis was put forward highlighting a role of arginine CGA codons together with glutamine CAA and CAG codons in the control of mRNA quality and life span. According to this hypothesis, the conversion of the ribonucleic codons CGA, CAA, or CAG into stop codons UGA, UAA or UAG owing to spontaneous or enzymatic cytosine deamination might serve as a trigger for the transcript destruction by NMD (C→U control). Thus, the consequences of epigenetic-mediated nonsense mutations are diverse and may largely depend on the structure of the transcript (CGA codon position, the presence and position of introns and SECIS elements, and splicing potential) of the cognate gene. However, this diversity and the presumable role of CGA codons in performing the everyday function by controlling whether genes are expressed correctly do not exclude their long-term role as limiters of the cell and organism life span. Thus, the presumable role of CGA codons in genome functioning and stability opens new perspectives to influence aging and concomitant deceases by codon editing.

James A. Shapiro: Evolution: a view from the twenty-first century

James A. Shapiro: Evolution: a view from the twenty-first century

Experimental evidence for Tyr171 involvement in catalytic cleavage by apurinic/apyrimidinic endonuclease (AP endo)

Abasic (AP) sites in DNA are highly mutagenic and as such must be efficiently repaired. AP endo (HAP1, Apex, APE1), the enzyme primarily responsible for recognizing AP sites in higher eukaryotic DNA, nicks the DNA backbone 5′ to the AP site. We have proposed that the phenolate form of Tyr171 in the active site is responsible for cleavage of AP DNA. In this work, we provide physical evidence for our hypothesis by comparing NMR spectra of WT and Y171F mutant proteins in the presence and absence of Mg2+ and AP DNA. Circular dichroism studies show that WT and Y171F proteins have comparable secondary structure. Y171F binds AP DNA with a Kd that is 300 times greater that that of WT. Despite this relatively small change in binding capacity, the catalytic efficiency is reduced by 4 orders of magnitude. Recombinant proteins were labeled at the amide nitrogen of all 14 tyrosines by growth in rich defined medium containing 15[N] tyrosine and all other 19 amino acids. Comparison of 1H-15N HSQC-NMR spectra of the WT and Y171F mutant enabled us to identify a single peak corresponding to Tyr171. Upon addition of AP site DNA in the presence of 4 mM EDTA, conditions where AP DNA binds enzyme but is not cleaved, the Tyr171 peak disappears. Taken together, these data support the proposal that AP endo catalyzes AP site cleavage by a unique mechanism involving tyrosine at the active site. Supported by NIH CA72702 (to PRS) and NIH CA98232 (to CdS)

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.

Natural ageing in the rat liver correlates with progressive stabilisation of DNA–nuclear matrix interactions and withdrawal of genes from the nuclear substructure

In the interphase nucleus, the DNA of higher eukaryotes is organised in supercoiled loops anchored to a nuclear matrix (NM). Replication, transcription and splicing seem to occur at macromolecular complexes organised upon the NM. Thus, the topological relationship between genes located in the loops and the NM appears to be very important for nuclear physiology. Here, we report that natural ageing in the rat liver correlates with a progressive strengthening of the NM framework and the stabilisation of the DNA loop–NM interactions, as well as with a progressive increase in the relative distance of genes to the NM. Both phenomena correlate with the gradual loss of proliferating potential and progression towards terminal differentiation in the hepatocytes, suggesting that wholesale modifications in the topological relationships within the cell nucleus are markers of tissue ageing and senescence, at least in the mammalian liver. We discuss the possible functional implications of such structural modifications that may underlie both terminal hepatocyte differentiation and their eventual replicative senescence.

Large-scale methylation analysis of human genomic DNA reveals tissue-specific differences between the methylation profiles of genes and pseudogenes.

Cytosine in CpG dinucleotides is frequently found to be methylated in the DNA of higher eukaryotes and differential methylation has been proposed to be a key element in the organization of gene expression in man. To address this question systematically, we used bisulfite genomic sequencing to study the methylation patterns of three X-linked genes and one autosomal pseudogene in two adult individuals and across nine different tissues. Two of the genes, SLC6A8 and MSSK1, are tissue-specifically expressed. CDM is expressed ubiquitously. The pseudogene, psi SLC6A8, is exclusively expressed in the testis. The promoter regions of the SLC6A8, MSSK1 and CDM genes were found to be essentially unmethylated in all tissues, regardless of their relative expression level. In contrast, the pseudogene psi SLC6A8 shows high methylation of the CpG islands in all somatic tissues but complete demethylation in testis. Methylation profiles in different tissues are similar in shape but not identical. The data for the two investigated individuals suggest that methylation profiles of individual genes are tissue specific. Taken together, our findings support a model in which the bodies of the genes are predominantly methylated and thus insulated from the interaction with DNA-binding proteins. Only unmethylated promoter regions are accessible for binding and interaction. Based on this model we propose to use DNA methylation studies in conjunction with large-scale sequencing approaches as a tool for the prediction of cis-acting genomic regions, for the identification of cryptic and potentially active CpG islands and for the preliminary distinction of genes and pseudogenes.

The purification of a mismatch-specific thymine-DNA glycosylase from HeLa cells.

G/T mispairs that arise in the DNA of higher eukaryotes as a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine must be corrected to G/C pairs. We describe here the purification to apparent homogeneity of the enzyme that initiates this repair process by excising the mispaired thymine from the hetero-duplex to generate an apyrimidinic site. The enzymatic activity could be attributed to a 55-kDa polypeptide, which was purified from extracts of HeLa cells by a combination of conventional and DNA-affinity chromatography. The enzyme is a mismatch-specific thymine-DNA N-glycosylase, capable of hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of the DNA and a mispaired thymine. In addition to the G/T, the enzyme can remove thymine also from C/T and T/T mispairs in the order G/T >> C/T > T/T. It has no detectable endonucleolytic activity on apyrimidinic sites and does not catalyze the removal of thymine from A/T pairs or from single-stranded DNA.

A dictionary of transcription control sequences

It is very difficult to distinguish control elements in the DNA of higher eukaryotes, since they are mingled with vast amounts of non-functional sequence. In contrast, protein-coding exons are much easier to analyse. Exons are often extensive and compact. Exon regions can be searched for open reading frames and compared with peptide and cDNA sequences. Of control elements, only promoters, in fact only the TATA box and other elements that have defined locations relative to the transcription initiation site, can be characterized by their location.

Understanding the molecular mechanism by which methylation influences gene expression

CpG dinucleotides that are found in the DNA of higher eukaryotes often contain cytosine that has been modified by methylation at the C5 position. The frequency with which 5-methyl cytosine is found at a particular site varies between cell types. Many studies have shown that the transcriptional control regions of genes that are expressed in a given cell type tend to be undermethylated, relative to the same sequences in other cell types. These observations support the idea that methylation may be involved in tissue-specific regulation of gene expression (recently reviewed in Refs 1, 2).

Dna Methylation in Eukaryote

The DNA of higher eukaryotes contains one minor base, namely 5-methylcytosine. The distribution of this minor base between different species and different DNA fractions will be considered together with the actual sequences methylated. The properties of the enzyme responsible for DNA modification will be reviewed, particular note being paid to the efficiency of methylation of different DNA substrates. Various possible functions of the 5-methylcytosine in DNA will be considered and particular attention will be paid to the finding that specific modified bases present in DNA not undergoing transcription are absent in the same genes when these are being actively transcribed.

5-Methylcytosine in eukaryotic DNA.

A small portion of the cytosine residues in the DNA of higher eukaryotes as well as in that of many lowe eukaryotes if methylated. The resulting 5-methylcytosine residues occur in specific in the DNA, usually adjacent to guanine residues on the 3' side. This methylation of eukaryotic DNA has been proposed to function in many ways, including control of transcription, maintenance of chromosome structure, repair of DNA, establishment of preferred sites for mutation, oncogenic transformation, and, in certain systems, protection of DNA against enzymatic degradation.

Specific DNA methylation sites in the vicinity of the chicken beta-globin genes.

METHYLATION of cytosine is the only post-synthetic modification so far detected in the DNA of higher eukaryotes and thus has been made the basis of several proposed mechanisms of gene activity and cellular differentiation1–3. All evidence indicates, however, that the overall content of 5-methylcytosine in DNA does not vary significantly between different tissues of the same organism and does not change throughout at least some steps of differentiation4–6. As 5-methylcytosine occurs predominantly in the dinucleotide sequence CpG (ref. 7) and as a number of bacterial restriction endonucleases can distinguish whether this sequence is methylated or unmethylated, it is now possible to investigate DNA methylation patterns in the vicinity of single genes8. In this report, we use this technique to provide a correlation between DNA methylation and the activity of the chicken β-globin genes.